A genus of plant pathogenic bacteria is the genus Agrobacteria. Agrobacterium bacteria are naturally occurring plant genetic engineers. Members of the Agrobacterium genus natively have the ability to transfer a segment of DNA from a plasmid hosted by the bacterium into the genome of a cell of a living plant. The DNA transferred into the plant (the T-DNA) causes the plant cells to initiate two activities. One activity is to manufacture a class of chemicals, called opines, which can be metabolized by the bacteria as a food source. The other activity is to initiate the growth of a tumorous mass referred to as a crown gall.
Agrobacterium initiates crown gall growth in order to create a microenvironment, the crown gall, where the bacteria can thrive and multiply. Unfortunately, the crown gall itself saps metabolic energy from the plant that would otherwise be directed at producing vegetative growth or fruit, thus reducing the yield that would otherwise be produced by that plant. In some perennial plant species where the plant is long-lived, such as grapes, stonefruits and roses, the impact of crown gall disease can be significant. For example, strains of the bacteria Agrobacterium vitis, which attacks vines, are the primary cause of grapevine crown gall, which is the most economically damaging bacterial disease of grape worldwide.
A number of different approaches exist for controlling plant diseases in commercial agriculture. One approach is based upon the application to plants or soil of chemical agents toxic or inimical to the disease-causing organism. A second approach is based on the development of plant varieties that are resistant to infection by the particular disease or strain of disease. One other approach is based on the use of a biological organism to control the disease-causing organism. This latter strategy is referred to as biocontrol.
Biocontrol agents control plant disease by secreting chemicals that act to inhibit or kill the disease-causing organism, or by simply occupying the ecological niche that would otherwise be available to the disease-causing organism. Some microorganisms are capable of inhibiting the growth of competing microbial strains through the use of toxins. If a bacterium can emit a broad-spectrum anti-bacterial toxin into its local environment, then that bacterium will have less competition in its ecological niche. As a result, many bacteria, and other microorganisms, have evolved genes for toxins. Often the plasmids that carry the genes encoding these toxins also carry genes conferring on its host immunity to that particular toxin. This is advantageous since obviously the toxin-secreting organism must have some mechanism to avoid the toxicity of its own toxin if the organism is to successfully populate the ecological niche it has cleared for itself.
Effective strategies to control crown gall by using biocontrol agents to control Agrobacterium growth are now known to exist. One problem associated with combating crown gall disease, however, is that different Agrobacterium species normally inhabit and attack plants in differing ecological micro-zones of the plant, as well as different plant species. For example, Agrobacterium rhizogenes normally lives in the root environment (the rhizosphere) of plants and attacks plant roots, while Agrobacterium tumefaciens normally attacks and infects plant stems or crowns. As a result, biocontrol strategies for crown gall disease must be focused on the micro-environment of the particular plant species and strains of Agrobacterium sought to be suppressed.
Agrobacterium rhizogenes strain K84, for example, is the most studied crown gall biological control strain and is commercially utilized for disease control on stone fruits worldwide. Strain K84 biological control is thought to be primarily due to the production of two plasmid-encoded antibiotics, agrocins 84 and 434, encoded by genes on pAgK84 and pAgK434 respectively, each of which accounts for a portion of the observed disease control. Agrocin 84, an adenine analog, is effective against tumorigenic strains carrying nopaline/agrocinopine pTi plasmids, and requires the accC gene in the target strain for activity. Agrocin 434, a di-substituted cytidine analog, is effective against, and specific for, a broad range of A. rhizogenes strains. The commercial application of the K84 biological control system, however, is limited to stone fruits as pathogenic Agrobacterium strains of other crops are not inhibited by K84.
With respect to the Agrobacterium vitis strains, the causative agents of grapevine crown gall, no effective preventative control measures are commercially available. One prior attempt to use a biocontrol technique to combat crown gall in grape was based on the Agrobacterium vitis bacterial strain F2/5. Strain F2/5 is a non-tumorigenic Agrobacterium strain which may be applied to grapevines to occupy the ecological niches that might otherwise be occupied by tumorigenic strains. This strategy has met with some success, but the success is both grape-specific, i.e. does not work on other plant species, and is variable depending on the identity of the virulent A. vitis strain causing the disease. For example, F2/5 is ineffective on non-grapevine host plants, such as Nicotiana glauca, sunflower or tomato, and ineffective against various pathogenic A. vitis strains, such as CG78, as well as other A. tumefaciens biovars.
What is needed is a biocontrol agent with a broader target range to help control crown gall disease and, in particular, crown gall disease in grape plants.